Nicola J. Smith

International Union of Pharmacology. LXXI. Free Fatty Acid Receptors FFA1, -2, and -3: Pharmacology and Pathophysiological Functions

Identification of G protein-coupled receptors that are activated by free fatty acids has led to considerable interest in their pharmacology and function because of the wide range of normal physiology and disease states in which fatty acids have been implicated. Free fatty acid receptor (FFA) 1 is activated by medium- to long-chain fatty acids and is expressed in the insulin-producing β-cells of the pancreas. Activation of FFA1 has been proposed to mediate fatty acid augmentation of glucose-stimulated insulin secretion although it is unclear whether the known long-term detrimental effects of β-cell exposure to high levels of fatty acids are also mediated through this receptor. The related receptors FFA2 and FFA3 are both activated by short-chain fatty acids although they have key differences in the signaling pathways they activate and tissue expression pattern. The aim of this review is to provide a comprehensive overview of the current understanding of the pharmacology and physiological role of these fatty acid receptors.

Allostery at G Protein-Coupled Receptor Homo- and Heteromers: Uncharted Pharmacological Landscapes

For many years seven transmembrane domain G protein-coupled receptors (GPCRs) were thought to exist and function exclusively as monomeric units. However, evidence both from native cells and heterologous expression systems has demonstrated that GPCRs can both traffic and signal within higher-order complexes. As for other protein-protein interactions, conformational changes in one polypeptide, including those resulting from binding of pharmacological ligands, have the capacity to alter the conformation and therefore the response of the interacting protein(s), a process known as allosterism. For GPCRs, allosterism across homo- or heteromers, whether dimers or higher-order oligomers, represents an additional topographical landscape that must now be considered pharmacologically. Such effects may offer the opportunity for novel therapeutic approaches. Allosterism at GPCR heteromers is particularly exciting in that it offers additional scope to provide receptor subtype selectivity and tissue specificity as well as fine-tuning of receptor signal strength. Herein, we introduce the concept of allosterism at both GPCR homomers and heteromers and discuss the various questions that must be addressed before significant advances can be made in drug discovery at these GPCR complexes.

Low Affinity GPCRs for Metabolic Intermediates: Challenges for Pharmacologists

The discovery that a number of metabolites and metabolic intermediates can act through G protein-coupled receptors has attracted great interest in the field and has led to new therapeutic targets for diseases such as hypertension, type 2 diabetes, inflammation, and metabolic syndrome. However, the low apparent affinity of these ligands for their cognate receptors poses a number of challenges for pharmacologists interested in investigating receptor structure, function or physiology. Furthermore, the endogenous ligands matched to their receptors have other, well established metabolic roles and thus selectivity is difficult to achieve. This review discusses some of the issues researchers face when working with these receptors and highlights the ways in which a number of these obstacles have been overcome.

Selective Orthosteric Free Fatty Acid Receptor 2 (FFA2) Agonists IDENTIFICATION OF THE STRUCTURAL AND CHEMICAL REQUIREMENTS FOR SELECTIVE ACTIVATION OF FFA2 VERSUS FFA3

Free fatty acid receptor 2 (FFA2; GPR43) is a G protein-coupled seven-transmembrane receptor for short-chain fatty acids (SCFAs) that is implicated in inflammatory and metabolic disorders. The SCFA propionate has close to optimal ligand efficiency for FFA2 and can hence be considered as highly potent given its size. Propionate, however, does not discriminate between FFA2 and the closely related receptor FFA3 (GPR41). To identify FFA2-selective ligands and understand the molecular basis for FFA2 selectivity, a targeted library of small carboxylic acids was examined using holistic, label-free dynamic mass redistribution technology for primary screening and the receptor-proximal G protein [35S]guanosine 5′-(3-O-thio)triphosphate activation, inositol phosphate, and cAMP accumulation assays for hit confirmation. Structure-activity relationship analysis allowed formulation of a general rule to predict selectivity for small carboxylic acids at the orthosteric binding site where ligands with substituted sp3-hybridized α-carbons preferentially activate FFA3, whereas ligands with sp2- or sp-hybridized α-carbons prefer FFA2. The orthosteric binding mode was verified by site-directed mutagenesis: replacement of orthosteric site arginine residues by alanine in FFA2 prevented ligand binding, and molecular modeling predicted the detailed mode of binding. Based on this, selective mutation of three residues to their non-conserved counterparts in FFA3 was sufficient to transfer FFA3 selectivity to FFA2. Thus, selective activation of FFA2 via the orthosteric site is achievable with rather small ligands, a finding with significant implications for the rational design of therapeutic compounds selectively targeting the SCFA receptors.

The Action and Mode of Binding of Thiazolidinedione Ligands at Free Fatty Acid Receptor 1

The endogenous ligands for free fatty acid receptor 1 (FFA1) are medium and longer chain free fatty acids. However, a range of selective, small molecule ligands have recently been developed as tool compounds to explore the therapeutic potential of this receptor, whereas clinically employed thiazolidinedione “glitazone” drugs are also agonists at FFA1. Each of these classes of agonist was able to promote phosphorylation of the ERK1/2 mitogen-activated protein (MAP) kinases in cells able to express human FFA1 on demand. However, although both lauric acid and the synthetic agonist GW9508X produced rapid and transient ERK1/2 MAP kinase phosphorylation, the thiazolidinedione rosiglitazone produced responses that were sustained for a substantially longer period. Despite this difference, the effects of each ligand required FFA1 and were transduced in each case predominantly via G proteins of the Gαq/Gα11 family. Different glitazone drugs also displayed markedly different efficacy and kinetics of sustainability of ERK1/2 MAP kinase phosphorylation. A number of orthosteric binding site mutants of FFA1 were generated, and despite variations in the changes of potency and efficacy of the three ligand classes in different functional end point assays, these were consistent with rosiglitazone also binding at the orthosteric site. Four distinct polymorphic variants of human FFA1 have been described. Despite previous indications that these display differences in function and pharmacology, they all responded in entirely equivalent ways to lauric acid, rosiglitazone, and GW9508X in measures of ERK1/2 MAP kinase phosphorylation, enhancement of binding of [35S]GTPγS (guanosine 5′-O-(3-[35S]thio)triphosphate) to Gαq, and elevation of intracellular [Ca2+], suggesting that individuals expressing each variant are likely to respond equivalently to orthosteric agonists of FFA1.

Conserved Polar Residues in Transmembrane Domains V, VI, and VII of Free Fatty Acid Receptor 2 and Free Fatty Acid Receptor 3 Are Required for the Binding and Function of Short Chain Fatty Acids

FFA2 and FFA3 are closely related G protein-coupled receptors that bind and respond to short chain fatty acids. (FFA2 and FFA3 are the provisional International Union of Pharmacology designations for the receptors previously called GPR43 and GPR41, respectively.) Sequence comparisons between these two receptors and alignments with the related G protein-coupled receptor FFA1, linked to homology modeling based on the atomic level structure of bovine rhodopsin, indicated the potential for polar residues within the transmembrane helix bundle to play important roles in ligand recognition and function. In both FFA2 and FFA3, mutation of either an arginine at the top of transmembrane domain V or a second arginine at the top of transmembrane domain VII eliminated the function of a range of short chain fatty acids. Mutation of a histidine in transmembrane domain VI, predicted to be in proximity to both the arginine residues, also eliminated function in many but not all assay formats. By contrast, mutation of a histidine in transmembrane domain IV, predicted to be lower in the binding pocket, modulated function in some assays of FFA3 function but had limited effects on the function of acetate and propionate at FFA2. Interestingly, wild type FFA3 responded to caproate, whereas FFA2 did not. Mutation of the transmembrane domain IV histidine eliminated responses of FFA3 to caproate but resulted in a gain of function of FFA2 to this six-carbon fatty acid. These data demonstrate the importance of positively charged residues in the recognition and/or function of short chain fatty acids in both FFA2 and FFA3. The development of small molecule ligands that interact selectively with these receptors will allow further details of the binding pockets to be elucidated.

Signal Switching, Crosstalk, and Arrestin Scaffolds: Novel G Protein–Coupled Receptor Signaling in Cardiovascular Disease

Hormone agonists, including angiotensin II (Ang II), norepinephrine, urotensin II, endothelin-1, vasopressin, and serotonin, mediate a plethora of physiological and pathological cardiovascular events via their cognate 7 membrane-spanning G protein–coupled receptors (GPCRs). On ligand binding, GPCRs undergo conformational changes that enable the activation and dissociation of heterotrimeric guanine nucleotide binding proteins (G proteins) and trigger a range of intracellular second messenger signaling cascades. Because of the immediacy of second messenger generation, GPCRs are able to acutely regulate cardiovascular events such as heart rate, contractile force, and systemic vascular resistance. Compelling evidence for GPCR control in the vasculature comes from both transgenic studies and clinical findings. For example, β-adrenergic receptor blockers and inhibitors of the synthesis and binding of Ang II are proven antihypertensive therapeutics for humans. Furthermore, mice lacking RGS2, a regulatory protein that enhances the speed of GPCR signal termination, display marked hypertension, increased basal vascular tone, and hypersensitivity to vasoconstrictive agonists,1 a phenotype that demonstrates the contribution of immediate GPCR-dependent signals, as well as the effect of GPCR dysregulation on cardiovascular homeostasis. The evidence that signals emanating from GPCRs also contribute to the chronic development of vascular disease is persuasive. Overexpression of the Gαq subunit in cardiomyocytes directly stimulates cardiac hypertrophy and decompensated heart failure,2 whereas transgenic mice expressing an inhibitory fragment of Gαq exhibit reduced hypertrophy in response to pressure overload.3 GPCR agonists like Ang II, endothelin-1, and norepinephrine, act directly on cardiomyocytes to stimulate hypertrophy.4 Meanwhile, Ang II contributes to atherosclerosis via activation of vascular smooth muscle cell (VSMC) migration and hypertrophy; this occurs either directly, via the Ang II type 1 receptor (AT1R), or indirectly by stimulating endothelin-1 expression or activating inflammatory pathways.5 Importantly, these pathological vascular effects require significant modulation of gene transcription, often via activation of …

Angiotensin1‐9 antagonises pro‐hypertrophic signalling in cardiomyocytes via the angiotensin type 2 receptor

Non‐technical summary The renin–angiotensin system regulates the function of the cardiovascular system via the peptide hormone angiotensin II through a cellular receptor, the angiotensin type 1 receptor. Angiotensin II can become overactive and contribute to the development of cardiovascular disease, resulting in hypertension and other disorders such as enlargement of the heart muscle cells, termed cardiomyocyte hypertrophy. Here we demonstrate, for the first time, that an alternative angiotensin peptide, called angiotensin 1‐9, is able to inhibit cardiomyocyte hypertrophy induced by angiotensin II by binding and activating an alternative angiotensin receptor, the type 2 receptor. This has implications for our understanding of the renin–angiotensin system in normal cardiovascular function and in cardiovascular disease.

Extracellular Loop 2 Of The Free Fatty Acid Receptor 2 Mediates Allosterism Of A Phenylacetamide Ago-Allosteric Modulator

Allosteric agonists are powerful tools for exploring the pharmacology of closely related G protein-coupled receptors that have nonselective endogenous ligands, such as the short chain fatty acids at free fatty acid receptors 2 and 3 (FFA2/GPR43 and FFA3/GPR41, respectively). We explored the molecular mechanisms mediating the activity of 4-chloro-α-(1-methylethyl)-N-2-thiazolylbenzeneacetamide (4-CMTB), a recently described phenylacetamide allosteric agonist and allosteric modulator of endogenous ligand function at human FFA2, by combining our previous knowledge of the orthosteric binding site with targeted examination of 4-CMTB structure-activity relationships and mutagenesis and chimeric receptor generation. Here we show that 4-CMTB is a selective agonist for FFA2 that binds to a site distinct from the orthosteric site of the receptor. Ligand structure-activity relationship studies indicated that the N-thiazolyl amide is likely to provide hydrogen bond donor/acceptor interactions with the receptor. Substitution at Leu173 or the exchange of the entire extracellular loop 2 of FFA2 with that of FFA3 was sufficient to reduce or ablate, respectively, allosteric communication between the endogenous and allosteric agonists. Thus, we conclude that extracellular loop 2 of human FFA2 is required for transduction of cooperative signaling between the orthosteric and an as-yet-undefined allosteric binding site of the FFA2 receptor that is occupied by 4-CMTB.

Hijacking epidermal growth factor receptors by angiotensin II: new possibilities for understanding and treating cardiac hypertrophy.

Abstract.Activation of the type 1 angiotensin II receptor (AT1R) is associated with the aetiology of left ventricular hypertrophy, although the exact intracellular signalling mechanism(s) remain unclear. Transactivation of the epidermal growth factor receptor (EGFR) has emerged as a central mechanism by which the G protein-coupled AT1R, which lacks intrinsic tyrosine kinase activity, can stimulate the mitogen-activated protein kinase signalling pathways thought to mediate cardiac hypertrophy. Current studies support a model whereby AT1R-dependent transactivation of EGFRs on cardiomyocytes involves stimulation of membrane-bound metalloproteases, which in turn cleave EGFR ligands such as heparin-binding EGF from a plasma membrane-associated precursor. Numerous aspects of the ‘triple membrane-passing signalling’ paradigm of AT1R-induced EGFR transactivation remain to be characterised, including the identity of the specific metalloproteases involved, the intracellular mechanism for their activation and the exact EGFR subtypes required. Here we examine how ‘hijacking’ of the EGFR might explain the ability of the AT1R to elicit the temporally and qualitatively diverse responses characteristic of the hypertrophic phenotype, and discuss the ramifications of delineating these pathways for the development of new therapeutic strategies to combat cardiac hypertrophy.